Effective therapies are needed to control excessive bleeding in a range of clinical conditions where bleeding cannot be adequately controlled by medical or surgical intervention. This unmet need is particularly critical among patients with hemophilia, especially those in whom factor replacement therapy is rendered less efficacious due to the production of inhibitor antibodies.
Activated clotting Factor X (FXa) occupies a central position in the coagulation cascade at the convergence of the intrinsic and extrinsic coagulation pathways. Membrane-bound FXa, in the presence of its cofactor Factor Va (FVa), converts prothrombin to thrombin, which activates platelets and converts fibrinogen to fibrin to form the thrombus. In principle, replacement therapy with direct FXa administration could correct bleeding. The therapeutic potential of FXa is limited, however, due to its very short plasma half-life and potential for inducing excessive coagulation due to activation of other coagulation factors.
Earlier work identified a FXa variant (I16L variant) in which leucine replaced isoleucine at the amino-terminus of the wild-type FXa heavy chain (position 16 in the chymotrypsin numbering scheme). The substitution yielded a FXa variant with zymogen-like characteristics. Toso, R., et al., The conformational switch from the factor X zymogen to protease state mediates exosite expression and prothrombinase assembly. J. Biol. Chem. 283, 18627-18635 (2008); Ivanciu L., et al., A zymogen-like factor Xa variant corrects the coagulation defect in hemophilia. Nat. Biotechnol. 29:1028-33 (2011).
When not incorporated into the prothrombinase complex with its cofactor Factor Va (FVa), the FXa I16L variant had no significant catalytic activity and was better protected from inactivation by serum protease inhibitors compared to wild-type FXa. As a result, the variant had longer serum half-life compared to wild-type FXa. Binding to FVa in prothrombinase, however, caused the variant to transition from the zymogen-like state to the active conformation, thereby restoring the ability of the variant to catalyze conversion of prothrombin to thrombin and thus its pro-coagulant activity. In mouse models of hemophilia A and hemophilia B, administering the FXa I16L variant before injury reduced blood loss following tail-clipping in a dose dependent manner. The results of these experiments suggest that the FXa I16L variant might be useful to treat uncontrolled bleeding in humans with hemophilia.
The FXa I16L variant used in the earlier studies, however, was made in small quantities from stably transfected HEK 293 cells followed by activation of the FX protein using Russell's viper venom protease (abbreviated “RVVX”). Toso, R., Zhu, H. & Camire, R. M. The conformational switch from the factor X zymogen to protease state mediates exosite expression and prothrombinase assembly. J. Biol. Chem. 283, 18627-18635 (2008). While useful for small-scale studies, this approach is not suitable for production of large quantities of purified FXa variant protein required for clinical studies and eventual supply to patients. Accordingly, there is a need in the art for preparations of the FXa I16L variant protein made in such quantity and purity that they may be tested in clinical trials, and once approved, provided to subjects in need of hemostasis.